This invention relates to antiviral agents and methods of use thereof. More particularly, the invention relates to antiviral agents that specifically destroy cells infected by viruses that produce a protease in such infected cells. The antiviral agents are activated by the viral protease, thereby specifically targeting the infected cells for destruction. Toxins that target cell surface receptors or antigens on tumor cells have attracted considerable attention for treatment of cancer. E.g., I. Pastan & D. FitzGerald, Recombinant Toxins for Cancer Treatment, 254 Science 1173–1177 (1991); Anderson et al., U.S. Pat. Nos. 5,169,933 and 5,135,736; Thorpe et al., U.S. Pat. No. 5,165,923; Jansen et al., U.S. Pat. No. 4,906,469; Frankel, U.S. Pat. No. 4,962,188; Uhr et al., U.S. Pat. No. 4,792,447; Masuho et al., U.S. Pat. Nos. 4,450,154 and 4,350,626. These agents include a cell-targeting moiety, such as an antigen-binding protein or a growth factor, linked to a plant or bacterial toxin. They kill cells by mechanisms different from conventional chemotherapy, thus potentially reducing or eliminating cross resistance to conventional chemotherapeutic agents.
Ricin and other similar plant toxins, such as abrin, modeccin and viscumin, comprise two polypeptide chains (known as the A and B chains) linked by a disulfide bridge, one chain (the A chain) being primarily responsible for the cytotoxicity and the other chain (the B chain) having sites that enable the molecule to bind to cell surfaces. Such toxins are known as type II ribosome-inactivating proteins or RIPs. F. Stirpe et al., Ribosome-inactivating Proteins from Plants: Present Status and Future Prospects, 10 Biotechnology 405–412 (1992).
Ricin is produced in the plant Ricinus communis (commonly known as the castor bean plant) via a precursor protein known as “preproricin.” Preproricin comprises a single polypeptide chain that includes a leader sequence, the A chain, a linker peptide, and the B chain. The leader sequence is subsequently removed in the organism to yield proricin, which is then cleaved to eliminate the linker region such that the A and B chains remain connected only by a disulfide bond in the mature protein. The toxicity of ricintype toxins operates in three phases: (1) binding to the cell surface via the B chain; (2) penetration of at least the A chain into the cytosol via intracellular organelles, and (3) inhibition of protein synthesis through the A chain cleaving an essential adenine residue from ribosomal RNA. Thus, outside the cell separated A and B chains are essentially nontoxic, because the inherently toxic A chain lacks the ability to bind to cell surfaces and enter the cells in the absence of the B chain. Moreover, preproricin and proricin are also non-toxic, since the activity of the A chain is inhibited in these precursors. It is also known that in ricintype toxins the B chain binds to cell surfaces by virtue of galactose recognition sites, which react with glycoproteins or glycolipids exposed on the cell surface. It has been suggested that the toxicity of the ricin A chain might be exploited in antitumor therapy by replacing the indiscriminatelybinding B chain with a different targeting component having the ability to bind only to tumor cells. Thus, various immunotoxins have been prepared consisting of a conjugate of whole ricin or a separated ricin A chain and a tumorspecific monoclonal antibody or other targeting component. While previously described immunotoxins comprising ricin are generally suitable for their specific purposes, they possess certain inherent limitations that detract from their overall utility in treating viral infections. One problem with the known conjugates arises from a structural feature of the A chain from natural ricin. It is known that the natural ricin A chain becomes Nglycosylated during its synthesis, by enzymes present in Ricinus cells, and it is thought that the resulting sugar moieties are capable of nonspecific interactions with cell surfaces. Thus, it appears that the known A chain conjugates are capable of a certain amount of binding with non target cells, even in the absence of the natural B chain, thus increasing the toxicity of such immunotoxins toward non-target cells. To partially mitigate this problem, recombinant A chain that lacks carbohydrate residues has been produced in E. coli. S. H. Pincus & V. V. Tolstikov, Anti-Human Immunodeficiency Virus Immunoconjugates, 32 Adv. Pharmacol. 205–247 (1995). Another problem with many ricin immunoconjugates arises from the fact that the B chain seems to have an important secondary function in that it somehow assists in the intoxication process, apart from its primary function in binding the ricin molecule to the cell surfaces. This secondary function is lost if the B chain is replaced by a different targeting component, such as a monoclonal antibody. Some researchers have addressed this problem by covalent attachment of affinity reagents to the B chain such that the galactose binding sites are blocked. J. M. Lambert et al., An Immunotoxin Prepared with Blocked Ricin: a Natural Plant Toxin Adapted for Therapeutic Use, 51 Cancer Res. 6236–6242 (1991).
The aforementioned modifications of ricin seek to enhance binding specificity to the outer cell surface by immunotoxins and similar, targeted therapeutic agents. Since certain types of infected cells do not express infection-related surface antigens, such binding specificity represents an inherent limitation. S. H. Pincus & V. V. Tolstikov, supra. A targeting-independent agent with a well-defined toxin activation mechanism involving a viral protease would permit the use of nonspecific “targeting” (i.e., cell-binding) molecules, including sugar moieties and fully active ricin B chain. Therapeutic agents designed in this manner could eliminate a broader spectrum of infected cells, with potentially fewer undesirable side effects. Anti-HIV immunotoxins have been described that include antibodies linked to various toxic moieties via a peptide linker that includes a sequence cleavable by HIV protease. S. H. Pincus & V. V. Tolstikov, supra. In some cases, release of the toxic moiety by this protease may render it active, although the specific activation mechanism was not further defined. In the present invention, antibodies or segments thereof are only one of many potential targeting molecules for the therapeutic agents. Moreover, the activation mechanisms are clearly specified in the present invention. One such mechanism relies on protease-dependent cleavage at or near the natural protease activation site for a given toxin, not merely on release from a bulky “carrier” protein (i.e., antibody). S. H. Pincus & V. V. Tolstikov, supra. In the case of ricin, the natural site for cleavage by proteolytic activity in Ricinus is in a disulfide-circumscribed loop in which one cysteine resides on the A chain and the other resides on the B chain; cleavage yields A and B chains connected by a disulfide bond. Therefore, most embodiments of the present invention that involve ricin include an HIV-protease cleavage sequence fused in-frame to the C-terminus of A chain such that the natural cleavage site is replaced with the HIV-protease site in the disulfide-circumscribed loop. In these embodiments, at lease some minimal N-terminal sequence of B chain required to inhibit A chain activity is retained, such that activation requires proteolytic cleavage and reduction of the disulfide bond. In all remaining embodiments, the mechanism of activation involves cleavage of a peptide linker to A chain, thereby separating adenine-like moieties that are chemically attached to the linker. Separation of the adenine-like residues unblocks the active site of ricin and allows A chain activity. Further, the foregoing text describes preferred embodiments (i.e., full B chain functionality, sugar moieties) that are highly compatible with these activation mechanisms. Indeed, these preferred embodiments are not suggested by others. S. H. Pincus & V. V. Tolstikov, supra. Certain embodiments of the present invention comprise attachment of hydrophobic moieties for intracellular targeting to sites of viral protease activity, which is limited in the cytosol. The aforementioned immunotoxins do not possess this aspect of the invention. While attachment of hydrophobic fatty acids to ricin A chain has been presented in terms of enhancing translocation across cell membranes for hypothetical medical applications, a method for activating ricin was not presented. A. V. Kabanov et al., Fatty Acylation of Proteins for Translocation Across Cell Membrane, 1 Biomed. Sci. 33–36 (1990); V. Y. Alakhov et al., Increasing Cytostatic Effects of Ricin A Chain and Staphylococcus aureus Enterotoxin A Through In Vitro Hydrophobization with Fatty Acid Residues, 12 Biotechnol. Appl. Biochem. 94–98 (1990). Hydrophobization of ricin is likely to increase toxicity to non-target cells, even if cell-surface targeting moieties are attached. A separate, viral-protease-dependent mechanism for activating ricin (and similar toxins) would prevent nonspecific toxicity. The present invention combines such a mechanism with hydrophobization.
In view of the foregoing, it will be appreciated that providing an antiviral agent that is activated only in cells infected with a selected virus, is non-toxic in uninfected cells, and is targeted independently of infection-related antigens, would be a significant advancement in the art. Furthermore, the prior art teaches away from making the present invention because specific embodiments described herein have previously been described as deleterious (B chain activity). Moreover, the prior art fails to describe or suggest elements of the present invention (e.g., means for fatty acid attachment) in combination with a protease-dependent toxin activation mechanism.